Liposomes are microscopic particles that are made up of one or more lipid bilayers enclosing an internal compartment. Liposomes can be categorized into multilamellar vesicles, multivesicular liposomes, unilamellar vesicles and giant liposomes. Liposomes have been widely used as carriers for a variety of agents such as drugs, cosmetics, diagnostic reagents, and genetic material. Since liposomes consist of non-toxic lipids, they generally have low toxicity and therefore are useful in a variety of pharmaceutical applications. In particular, liposomes are useful for increasing the circulation lifetime of agents that have a short half-life in the bloodstream. Liposome encapsulated drugs often have biodistributions and toxicities which differ greatly from those of free drug. For specific in vivo delivery, the sizes, charges and surface properties of these carriers can be changed by varying the preparation methods and by tailoring the lipid makeup of the carrier. For instance, liposomes may be made to release a drug more quickly by decreasing the acyl chain length of a lipid making up the carrier.
The most efficient method of encapsulating a high drug payload in liposomes is via active loading. This process is mediated by the creation of pH gradients (ΔpH) or metal ion gradients (ΔM2+) across the liposomal membrane. For example, a ΔpH generated by preparing liposomes in citrate buffer pH 4.0 followed by exchange of external buffer with buffered-saline pH 7.5, can promote the liposomal accumulation of weakly basic drugs. The neutral form of the drug passively diffuses across the lipid bilayer and becomes trapped upon protonation in the low pH environment of the liposome interior. This process can result in >98% drug encapsulation and high drug-to-lipid ratios (e.g. vinorelbine). Drug loading via ΔM2+ follows an analogous process, with drug accumulation being driven by metal ion-complexation (e.g. doxorubicin-Mn2+). Drug loading efficiencies driven by metal ion-complexation are comparable to those described for ΔpH.
A further active loading procedure uses a combination of an ionophore, such as A23187, and divalent metal ions (M2+). A23187 incorporates into the lipid bilayer and exchanges 1M2+ from the interior liposome buffer for 2H+ from the external buffer, thereby generating and maintaining a ΔpH. A23187 and an internal Mn2+ buffer have been used previously to efficiently encapsulate vincristine, ciprofloxacin, topotecan and irinotecan. The role of Mn2+ in this system is believed to be an ‘inert’ facilitator for the creation of a ΔpH. Indeed, exchanging the internal Mn2+-based buffer with a Cu2+-based buffer does not result in any differences in the kinetics of anticancer drug loading.
The present invention relates to the use of divalent copper ions (Cu2+) to significantly enhance intra-liposomal drug retention attributes and hence in vivo therapeutic effects. For example, a Cu2+/A23187 liposomal irinotecan formulation described herein demonstrated significantly improved efficacy against murine xenograft models of colorectal cancer when compared to the Mn2+/A23187 equivalent. The Applicant's loading procedure combines high encapsulation efficiencies (>98%), high drug-to-lipid ratios and enhanced drug retention. It is notable that existing inventions describe the use of transmembrane pH gradients, defining the utility of ionophores to generate a transmembrane pH gradient, or alternatively disclose the use of transition metals, such as Mn2+ or Cu2+, in the presence of a neutral environment and in the absence of a transmembrane pH gradient (Fenske et al., U.S. Pat. No. 5,837,282; Tardi et al., US 20030091621). The prior art does not teach methods and compositions that rely on divalent copper ions (Cu2+) in a low pH environment, and it was not anticipated that such compositions would result in improved drug retention attributes.
Liposomes containing metal ions encapsulated in the interior of the vesicle have previously be used in diagnostic applications. For example, liposomes have been used for delivery of contrast agents with the goal of accumulating a contrast agent at a desired site within the body of a subject. In the latter application, liposomes have mainly been used for delivery of diagnostic radionucleotides and paramagnetic metal ions in gamma and magnetic resonance imaging, respectively. However, liposomally encapsulated metal ions in these applications are not employed for drug retention purposes.
Camptothecins are a class of anticancer drugs that inhibit the nuclear enzyme, topoisomerase I (topo I). Topo I facilitates DNA replication during the S phase of the cell cycle by inducing transient single strand breaks in the DNA double-helix. The complex formed between DNA and topo I is referred to as the ‘cleavable complex’. Camptothecins induce caspase-mediated cellular apoptosis by stabilising this cleavable complex. Camptothecins, such as irinotecan, possess a lactone ring. This lactone ring is crucial for cytotoxic activity. Liposome formulations of camptothecins are an attractive option based on the potential for these carrier systems to maintain the drug in an environment that favours the active closed ring lactone form. An equilibrium exists between the closed lactone ring form of camptothecins and an inactive open-ring carboxylic acid form (FIG. 1). This equilibrium is influenced by pH. At acidic pH, equilibrium is driven towards the closed lactone ring. At neutral or alkaline pH (e.g. physiological conditions), equilibrium favours the inactive open-ring form.
The need has arisen for improved liposomal formulations which both enhance retention of encapsulated drugs and also preferably maintain the drugs in their active form for improved delivery and efficacy at a target site.